scholarly journals Shear and Integrin Outside-In Signaling Activates NADPH-Oxidase 2 to Promote Platelet Activation

2021 ◽  
Author(s):  
Zheng Xu ◽  
Ying Liang ◽  
M. Keegan Delaney ◽  
Yaping Zhang ◽  
Kyungho Kim ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Platelet Activation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Regulatory Subunit ◽  
Ros Generation ◽  
Platelet Response ◽  
Nadph Oxidase 2 ◽  
Phosphoinositide 3 Kinase ◽  
Platelet Spreading

Objective: Despite the importance of reactive oxygen species (ROS) and NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) 2 in platelet activation and in vivo thrombosis, it is unclear how ROS and NOX2 play a role in platelet activation and why NOX2 deficiencies in humans and mice do not affect hemostasis. Outside-in signaling of integrin α IIb β 3 mediates platelet response to shear stress, secondary platelet activation, and thrombus expansion and is critical to thrombosis but dispensable for hemostasis. We studied the mechanisms of platelet ROS generation, ROS-mediated platelet response, and the role of ROS in integrin α IIb β 3 outside-in signaling. Approach and Results: ROS generation in activated platelets was low and slow without shear but was robust under shear. Shear-enhanced ROS generation and activation of p47phox, an important regulatory subunit of NOX2, were diminished by the integrin antagonist integrilin or β 3 knockout, and by Gα 13 knockout or blocking the Gα 13 -β 3 interaction. Resting platelets spreading on integrin ligand fibrinogen also Gα 13 -dependently stimulated ROS generation and p47phox activation. Hence, Gα 13 -mediated outside-in signaling induces NOX2 activation and ROS generation which is greatly enhanced by shear. Outside-in NOX2 activation requires Src, phosphoinositide 3-kinase and Akt downstream of Gα 13 . Importantly, NOX2-knockout platelets showed defective ROS generation, reduced platelet spreading without shear, and reduced platelet adhesion and thrombus volume on collagen and VWF (von Willibrand factor) under shear, whereas ROS inhibition diminished activation of tyrosine kinase Syk. Conclusions: Outside-in signaling activates the mainly NOX2-mediated ROS generation, which mediates Syk-dependent secondary platelet activation, adhesion, and thrombosis with minimal effect on hemostasis.

scholarly journals Different Degrees of NADPH Oxidase 2 Regulation and In Vivo Platelet Activation: Lesson From Chronic Granulomatous Disease

2014 ◽  
Vol 3 (3) ◽  
Author(s):  
Roberto Carnevale ◽  
Lorenzo Loffredo ◽  
Valerio Sanguigni ◽  
Alessandro Plebani ◽  
Paolo Rossi ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Platelet Activation ◽  
Granulomatous Disease ◽  
Nadph Oxidase 2

scholarly journals Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

2019 ◽  
Vol 20 (18) ◽  
pp. 4357 ◽  
Author(s):  
Ying-Ying Chen ◽  
Tsung-Tien Wu ◽  
Chiu-Yi Ho ◽  
Tung-Chen Yeh ◽  
Gwo-Ching Sun ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Nadph Oxidase ◽  
Sglt2 Inhibitor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reduced Form ◽  
Ros Generation ◽  
Protein Levels ◽  
Sodium Glucose Cotransporter ◽  
Patients With Diabetes

Purpose: Cataracts in patients with diabetes mellitus (DM) are a major cause of blindness in developed and developing countries. This study aims to examine whether the generation of reactive oxygen species (ROS) via the increased expression of glucose transporters (GLUTs) and the receptor for advanced glycation end products (RAGE) influences the cataract development in DM. Methods: Lens epithelial cells (LECs) were isolated during cataract surgery from patients without DM or with DM, but without diabetic retinopathy. In a rat model, fructose (10% fructose, 8 or 12 weeks) with or without dapagliflozin (1.2 mg/day, 2 weeks) treatment did induce DM, as verified by blood pressure and serum parameter measurements. Immunofluorescence stainings and immunoblottings were used to quantify the protein levels. Endogenous O2˙¯ production in the LECs was determined in vivo with dihydroethidium stainings. Results: We investigated that GLUT levels in LECs differed significantly, thus leading to the direct enhancement of RAGE-associated superoxide generation in DM patients with cataracts. Superoxide production was significantly higher in LECs from rats with fructose-induced type 2 DM, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin prevented this effect in fructose-fed rats. Protein expression levels of the sodium/glucose cotransporter 2 (SGLT2), GLUT1, GLUT5, the nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit p67-phox, NOX2/4 and RAGE were upregulated in fructose-fed animals, whereas dapagliflozin treatment reversed these effects. Conclusions: In rats with fructose-induced DM, dapagliflozin downregulates RAGE-induced NADPH oxidase expression in LECs via the inactivation of GLUTs and a reduction in ROS generation. These novel findings suggest that the SGLT2 inhibitor dapagliflozin may be a candidate for the pharmacological prevention of cataracts in patients with DM.

Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Michael K Delaney ◽  
Kyungho Kim ◽  
Brian Estevez ◽  
Aleksandra Stojanovic-Terpo ◽  
Bo Shen ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Platelet Activation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
General Role ◽  
Glycoprotein Vi ◽  
Activation Pathways

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

Cadmium Chloride Induces Memory Deficits and Hippocampal Damage by Activating the JNK/p66Shc/NADPH Oxidase Axis

2020 ◽  
Vol 39 (5) ◽  
pp. 477-490
Author(s):  
Attalla Farag El-kott ◽  
Ali S. Alshehri ◽  
Heba S. Khalifa ◽  
Abd-El-karim M. Abd-Lateif ◽  
Mohammad Ali Alshehri ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Cadmium Chloride ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Male Rats ◽  
Hippocampal Damage ◽  
Vitro Experiment ◽  
In Vitro Experiment ◽  
In Vivo Experiment

This study investigated whether the mechanism underlying the neurotoxic effects of cadmium chloride (CdCl2) in rats involves p66Shc. This study comprised an initial in vivo experiment followed by an in vitro experiment. For the in vivo experiment, male rats were orally administered saline (vehicle) or CdCl2 (0.05 mg/kg) for 30 days. Thereafter, spatial and retention memory of rats were tested and their hippocampi were used for biochemical and molecular analyses. For the in vitro experiment, control or p66Shc-deficient hippocampal cells were treated with CdCl2 (25 µM) in the presence or absence of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. Cadmium chloride impaired the spatial learning and retention memory of rats; depleted levels of glutathione and manganese superoxide dismutase; increased reactive oxygen species (ROS), tumor necrosis factor α, and interleukin 6; and induced nuclear factor kappa B activation. Cadmium chloride also decreased the number of pyramidal cells in the CA1 region and induced severe damage to the mitochondria and endoplasmic reticulum of cells in the hippocampi of rats. Moreover, CdCl2 increased the total unphosphorylated p66Shc, phosphorylated (Ser36) p66Shc, phosphorylated JNK, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, cytochrome c, and cleaved caspase-3. A dose–response increase in cell death, ROS, DNA damage, p66Shc, and NADPH oxidase was also observed in cultured hippocampal cells treated with CdCl2. Of note, all of these biochemical changes were attenuated by silencing p66Shc or inhibiting JNK with SP600125. In conclusion, CdCl2 induces hippocampal ROS generation and apoptosis by promoting the JNK-mediated activation of p66Shc.

scholarly journals NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease

2020 ◽  
Vol 4 (23) ◽  
pp. 5976-5987
Author(s):  
Suk See De Ravin ◽  
Julie Brault ◽  
Ronald J. Meis ◽  
Linhong Li ◽  
Narda Theobald ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Messenger Rna ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Functional Restoration ◽  
In Vivo Studies ◽  
Allogeneic Bone ◽  
Granulomatous Disease ◽  
Primate Model

Abstract Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation

2003 ◽  
Vol 90 (10) ◽  
pp. 672-678 ◽  
Author(s):  
Zhang Jian-ning ◽  
Angela Bergeron ◽  
Qinghua Yu ◽  
Carol Sun ◽  
Latresha McBride ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Fluid Shear Stress ◽  
High Shear ◽  
Platelet Response ◽  
Short Pulses ◽  
Hydrodynamic Effects

SummaryPlatelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent αIIbβ3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.

scholarly journals NADPH Oxidase 2 Mediates Myocardial Oxygen Wasting in Obesity

Antioxidants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 171 ◽  
Author(s):  
Anne D. Hafstad ◽  
Synne S. Hansen ◽  
Jim Lund ◽  
Celio X. C. Santos ◽  
Neoma T. Boardman ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Mitochondrial Function ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Mechanical Efficiency ◽  
Ros Production ◽  
Knock Out ◽  
Obesity And Diabetes ◽  
Mitochondrial Ros ◽  
Nadph Oxidase 2 ◽  
Knock Out Mice

Obesity and diabetes are independent risk factors for cardiovascular diseases, and they are associated with the development of a specific cardiomyopathy with elevated myocardial oxygen consumption (MVO2) and impaired cardiac efficiency. Although the pathophysiology of this cardiomyopathy is multifactorial and complex, reactive oxygen species (ROS) may play an important role. One of the major ROS-generating enzymes in the cardiomyocytes is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), and many potential systemic activators of NOX2 are elevated in obesity and diabetes. We hypothesized that NOX2 activity would influence cardiac energetics and/or the progression of ventricular dysfunction following obesity. Myocardial ROS content and mechanoenergetics were measured in the hearts from diet-induced-obese wild type (DIOWT) and global NOK2 knock-out mice (DIOKO) and in diet-induced obese C57BL/6J mice given normal water (DIO) or water supplemented with the NOX2-inhibitor apocynin (DIOAPO). Mitochondrial function and ROS production were also assessed in DIO and DIOAPO mice. This study demonstrated that ablation and pharmacological inhibition of NOX2 both improved mechanical efficiency and reduced MVO2 for non-mechanical cardiac work. Mitochondrial ROS production was also reduced following NOX2 inhibition, while cardiac mitochondrial function was not markedly altered by apocynin-treatment. Therefore, these results indicate a link between obesity-induced myocardial oxygen wasting, NOX2 activation, and mitochondrial ROS.

A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4156-4165 ◽  
Author(s):  
Catherine Trumel ◽  
Bernard Payrastre ◽  
Monique Plantavid ◽  
Béatrice Hechler ◽  
Cécile Viala ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Kinase Inhibitors ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Activation Process ◽  
Irreversible Aggregation ◽  
Stable Association ◽  
Phosphoinositide 3 Kinase

Abstract Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

scholarly journals Cytokine-dependent regulation of NADPH oxidase activity and the consequences for activated T cell homeostasis

2009 ◽  
Vol 206 (7) ◽  
pp. 1515-1523 ◽  
Author(s):  
Divya Purushothaman ◽  
Apurva Sarin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nadph Oxidase ◽  
Cell Apoptosis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Activated T Cells ◽  
T Cell Apoptosis ◽  
Mitochondrial Superoxide ◽  
Activated T Cell

Cellular dependence on growth factors for survival is developmentally programmed and continues in adult metazoans. Antigen-activated T cell apoptosis in the waning phase of the immune response is thought to be triggered by depletion of cytokines from the microenvironment. T cell apoptosis resulting from cytokine deprivation is mediated by reactive oxygen species (ROS), but their source and position in the apoptotic cascade is poorly understood. RNA interference approaches implicated the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neglect-induced apoptosis in T cells. Using mice deficient for the catalytic subunit gp91phox to characterize the molecular link to activated T cell apoptosis, we show that gp91phox-deficient T (T−/−) cells generated mitochondrial superoxide but had diminished hydrogen peroxide production in response to neglect, which, in turn, regulated Jun N-terminal kinase–dependent Bax activation and apoptosis. Activated T−/− cells were distinguished by improved survival after activation by superantigens in vivo, adoptive transfers into congenic hosts, and higher recall responses after immunization. Thus, the NADPH oxidase may regulate adaptive immunity in addition to its previously well-characterized role in the innate response.

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